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1.
SynerGraft® (SG) decellularized–cryopreserved cardiac valve allografts have been developed to provide a valve replacement option that has reduced antigenicity, retained structural integrity, and the ability to be stored long-term until needed for implantation. However, it is critical to ensure that both the SG processing and cryopreservation of these allografts do not detrimentally affect the extracellular matrix architecture within the tissue. This study evaluates the effects of SG decellularization and subsequent cryopreservation on the extracellular matrix integrity of allograft heart valves. Human aortic and pulmonary valves were trisected, with one-third of each either left fresh (no further processing after dissection), decellularized, or decellularized and cryopreserved. Two-photon laser scanning confocal microscopy was used to visualize collagen and elastin in leaflets and conduits. The optimized percent laser transmission (OPLT) required for full dynamic range imaging of each site was determined, and changes in OPLT were used to infer changes in collagen and elastin signal intensity. Collagen fiber crimp period and collagen and elastin fiber diameter were measured in leaflet tissue. Statistically significant differences in OPLT and the dimensional characteristics of collagen and elastin in study groups were determined through single factor ANOVA. The majority of donor-aggregated average OPLT observations showed no statistically significant differences among all groups, indicating no difference in collagen or elastin signal strength. Morphometric analysis of collagen and elastin fibers revealed no significant alterations in treated leaflet tissues relative to fresh tissues. Collagen and elastin structural integrity within allograft heart valves are maintained through SynerGraft® decellularization and subsequent cryopreservation.  相似文献   

2.
Recent advances in tissue engineering have led to potential new strategies, especially decellularization protocols from natural tissues, for the repair, replacement, and regeneration of intervertebral discs. This study aimed to validate our previously reported method for the decellularization of annulus fibrosus (AF) tissue and to quantify potentially antigenic α-Gal epitopes in the decellularized tissue. Porcine AF tissue was decellularized using different freeze–thaw temperatures, chemical detergents, and incubation times in order to determine the optimal method for cell removal. The integrity of the decellularized material was determined using biochemical and mechanical tests. The α-Gal epitope was quantified before and after decellularization. Decellularization with freeze–thaw in liquid nitrogen, an ionic detergent (0.1% SDS), and a 24 h incubation period yielded the greatest retention of GAG and collagen relative to DNA reduction when tested as single variables. Combined, these optimal decellularization conditions preserved more GAG while removing the same amount of DNA as the conditions used in our previous study. Components and biomechanical properties of the AF matrix were retained. The decellularized AF scaffold exhibited suitable immune-compatibility, as evidenced by successful in vivo remodeling and a decrease in the α-Gal epitope. Our study defined the optimal conditions for decellularization of porcine AF tissues while preserving the biological composition and mechanical properties of the scaffold. Under these conditions, immunocompatibility was evidenced by successful in vivo remodeling and reduction of the α-Gal epitope in the decellularized material. Decellularized AF scaffolds are potential candidates for clinical applications in spinal surgery.  相似文献   

3.
Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.  相似文献   

4.
Tissue-specific extracellular matrix plays an important role in promoting tissue regeneration and repair. We hypothesized that decellularized annular fibrosus matrix may be an appropriate scaffold for annular fibrosus tissue engineering. We aimed to determine the optimal decellularization method suitable for annular fibrosus. Annular fibrosus tissue was treated with 3 different protocols with Triton X-100, sodium dodecyl sulfate (SDS) and trypsin. After the decellularization process, we examined cell removal and preservation of the matrix components, microstructure and mechanical function with the treatments to determine which method is more efficient. All 3 protocols achieved decellularization; however, SDS or trypsin disturbed the structure of the annular fibrosus. All protocols maintained collagen content, but glycosaminoglycan content was lost to different degrees, with the highest content with TritonX-100 treatment. Furthermore, SDS decreased the tensile mechanical property of annular fibrosus as compared with the other 2 protocols. MTT assay revealed that the decellularized annular fibrosus was not cytotoxic. Annular fibrosus cells seeded into the scaffold showed good viability. The Triton X-100–treated annular fibrosus retained major extracellular matrix components after thorough cell removal and preserved the concentric lamellar structure and tensile mechanical properties. As well, it possessed favorable biocompatibility, so it may be a suitable candidate as a scaffold for annular fibrosus tissue engineering.  相似文献   

5.
Tissue engineering of heart valves utilizes biodegradable or metabolizable scaffolds for remodeling by seeded autologous cells. The aim of this study was to determine and compare extracellular matrix (ECM) formations, cellular phenotypes and cell location of native and tissue engineered (TE) valve leaflets. Ovine carotid arteries, ovine and porcine hearts were obtained from slaughterhouses. Cells were isolated from carotid arteries and dissected ovine, porcine and TE leaflets. TE constructs were fabricated from decellularized porcine pulmonary valves, seeded ovine arterial cells and subsequent 16 days dynamic in vitro culture using a pulsatile bioreactor. Native and TE valves were studied by histology (hematoxylin-eosin, resorcin-fuchsin, Movat pentachrome), NIR femtosecond multiphoton laser scanning microscopy and scanning electron microscopy (SEM). Cells of native and TE tissues were identified and localized by immunohistochemistry. Arterial, valvular and re-isolated TE-construct cells were processed for immunocytochemistry and Western blotting. ECM analysis and SEM revealed characteristical and comparable structures in native and TE leaflets. Most cells in native leaflets stained strongly positive for vimentin. Cells positive to alpha-smooth muscle actin (alpha-SMA), myosin and calponin were only found at the ventricular (inflow) side of ovine aortic and porcine pulmonary valve leaflets. Cells from TE constructs had a strong expression of vimentin, alpha-SMA, myosin, calponin and h-caldesmon throughout the entire leaflet. Comparable ECM formation and endothelial cell lining of native and TE leaflets could be demonstrated. However, immunostaining revealed significant differences between valvular cell phenotypes of native and TE leaflets. These results may be essential for further cardiovascular tissue engineering efforts.  相似文献   

6.
Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engineering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), sodium dodecyl sulfate (SDS) or sodium deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2 h and DS 2% for 1 h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. In addition, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering.  相似文献   

7.
The limited biocompatibility of decellularized scaffolds is an ongoing challenge in tissue engineering. Here, we demonstrate the residual immunogenicity of an extensively decellularized equine carotid artery (dEACintens) and identify the involved immunogenic components. EAC were submitted to an elaborated intensified decellularization protocol with SDS/sodium desoxycholate for 72 h using increased processing volumes (dEACintens), and compared to dEACord prepared by an ordinary protocol (40 h, normal volumes). Matrix integrity was checked via correlative volumetric visualization which revealed only minor structural changes in the arterial wall. In dEACintens, a substantial depletion of cellular components was obvious for smooth muscle actin (100%), MHC I complexes (97.8%), alphaGal epitops (98.4% and 91.3%) and for DNA (final concentration of 0.34±0.16 ng/mg tissue). However, dEACintens still evoked antibody formation in mice after immunization with dEACintens extracts, although to a lower extent than dEACord. Mouse plasma antibodies recognized a 140 kDa band which was revealed to contain collagen VI alpha1 and alpha2 chains via mass spectrometry of both 2D electrophoretically separated and immunoprecipitated proteins. Thus, even the complete removal of cellular proteins did not yield non-immunogenic dEAC as the extracellular matrix still conferred immunogenicity by collagen VI. However, as lower antibody levels were achieved by the intensified decellularization protocol, this seems to be a promising basis for further development.  相似文献   

8.
The effect of decellularization on the biomechanical properties of macaque lungs was studied. The quality of the biological scaffold was additionally assessed by morphological methods, and the contents of extracellular matrix (ECM) fibers were determined both qualitatively and quantitatively. Histological analysis revealed no damage of structural integrity of ECM components, but the scaffold elasticity significantly decreased, which was confirmed by the changes in the hysteresis loop without a concomitant decrease in peak loads, with the mechanical strength of the samples being retained. These changes require taking additional measures to prevent a decrease in the effective lung volume.  相似文献   

9.
Decellularized arteries have been considered as promising scaffolds for small-diameter vascular substitutes. However, weakened mechanical properties, immunological rejection and rapid degradation after transplantation still exist after decellularization. Previous studies indicated that genipin cross-linking can solve these problems. Therefore, genipin was selected as the cross-linking agent for the pre-treatment of decellularized arteries in our study. Histological analysis, scanning electron microscopy, mechanical properties analysis and subcutaneous embedding experiment were adopted to investigate the efficiency of decellularization and the effect of genipin cross-linking on improving mechanical, structural and biological properties of decellularized arteries. Decellularization protocols based on three trypsin concentrations were used to prepare decellularized arteries, after decellularization, arteries were cross-linked with genipin. Results showed that 0.5% trypsin was the most efficient concentration to remove cellular components and preserve ECM. However, mechanical properties of 0.5% trypsin decellularized arteries weakened significantly, while genipin cross-linking improved mechanical properties of decellularized arteries to the same level as fresh arteries. After 4 weeks subcutaneous embedding, cross-linked arteries caused the mildest inflammatory response. In conclusion, genipin could be employed as an ideal cross-linking agent to strengthen mechanical properties, enhance the resistance to degradation and reduce the antigenicity of decellularized arteries for small-diameter blood vessel tissue engineering applications.  相似文献   

10.
Decellularization, a technique used in liver regenerative medicine, is the removal of all the cellular components from a tissue or organ, leaving behind an intact structure of extracellular matrix. The biomechanical properties of this novel scaffold material are currently unknown and are important due to the mechanosensitivity of liver cells. Characterizing this material is important for bioengineering liver tissue from this decellularized scaffold as well as creating new 3-dimensional mimetic structures of liver extracellular matrix. This study set out to characterize the biomechanical properties of perfused liver tissue in its native and decellularized states on both a macro- and nano-scale. Poroviscoelastic finite element models were then used to extract the fluid and solid mechanical properties from the experimental data. Tissue-level spherical indentation-relaxation tests were performed on 5 native livers and 8 decellularized livers at two indentation rates and at multiple perfusion rates. Cellular-level spherical nanoindentation was performed on 2 native livers and 1 decellularized liver. Tissue-level results found native liver tissue to possess a long-term Young’s modulus of 10.5 kPa and decellularized tissue a modulus of 1.18 kPa. Cellular-level testing found native tissue to have a long-term Young’s modulus of 4.40 kPa and decellularized tissue to have a modulus of 0.91 kPa. These results are important for regenerative medicine and tissue engineering where cellular response is dependent on the mechanical properties of the engineered scaffold.  相似文献   

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